Biochemical factors governing the steady-state estrone/estradiol ratios catalyzed by human 17β-hydroxysteroid dehydrogenases types 1 and 2 in HEK-293 cells
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Human 17β-hydroxysteroid dehydrogenase types 1 and 2 (17βHSD1 and 17βHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17βHSD1 and 17βHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17βHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2′-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP+ ratio. The shift to E2 oxidation parallels changes in apparent Km values for purified 17βHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17βHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17βHSD2, and these mutations lower the apparent Km of recombinant 17βHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17βHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP+ ratios. In contrast, the preference of 17βHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17βHSD2 in intact cells.
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The directional preference of some reductive enzymes can be reduced or reversed by depleting cells of NADPH or by mutations that impair NADPH binding.
“Consequently, the directional preference of some”
From The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders (2011) · cites this paper as [53]
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The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders
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- Authors
- Sherbet, D. P., Guryev, O. L., Papari-Zareei, M., Mizrachi, D., Rambally, S., Akbar, S., & Auchus, R. J.
- Affiliations
- The University of Texas Southwestern Medical Center (US)
- Corresponding author
- Auchus, Richard J.
- Venue
- Endocrinology
- Publisher
- The Endocrine Society
- Published
- 2009-06-25
- Volume
- 150
- Issue
- 9
- Pages
- 4154-4162
- ISSN
- 0013-7227 (ISSN-L)
- 1945-7170
- PMC
- PMC2736091
- OpenAlex
- W2112297680
- Semantic Scholar
- 4e99665763f51d685d21bb12a1791b776339a950
- Cited by
- 22
- References
- 41
- Citations by year
- 2025: 1 · 2023: 1 · 2020: 1 · 2019: 3 · 2018: 1 · 2014: 4 · 2013: 1 · 2012: 3
- FWCI
- 2.13
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Endocrinology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Howard Hughes Medical Institute
- March of Dimes Foundation
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Estrogen and related hormone effects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
- Keywords
- 17β-hydroxysteroid dehydrogenase type 1, 17β-hydroxysteroid dehydrogenase type 2, estrone, estradiol, NADPH/NADP+ ratio, enzyme kinetics, site-directed mutagenesis, glucose deprivation, redox state, estrogen metabolism, HEK293 cells
- MeSH terms
- Amino Acid Sequence, Cell Line, Cloning, Molecular, Estradiol, Estradiol Dehydrogenases, Estrone, Humans, Kidney, Kinetics, Molecular Sequence Data, NADP, 17-Hydroxysteroid Dehydrogenases, Sequence Alignment, Amino Acid Substitution
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